Pullulan is a viscous water-soluble neutral polysaccharide having diverse applications. Its uses include: transparent films; oxygen-impermeable, tasteless, odorless coatings for food; viscosity control agents; adhesives for construction materials; fibers; and dielectrical materials in the form of cyanoethylpullulan. The purity of the polymer and molecular weight are important properties for the end uses.
Pullulan is an exopolysaccharide secreted by the fungus Aureobasidium pullulans (hereinafter referred to as A. pullulans) (Bernier, B., 1958, Canadian Journal of Microbiology 4:195-204; Bender, H. et al., 1959, Biochimica et Biophysica Acta 36:309-316). Several other similar isolates from nature also secrete pullulan (see comprehensive review by Catley, B. J., 1979, in Microbial Polysaccharides and Polysaccharases, R. C. W. Berkeley, G. W. Gooday and D. C. Ellwood [eds.] Academic Press).
A. pullulans is included in the American Type Culture Collection under one genus and species designation. The fungus is commonly isolated from forest litter, natural waters, industrial waste waters, paint surfaces, plastic, wood, leather, plant surfaces and canvass, and as an opportunistic pathogen from certain patients.
A. pullulans is a polymorphic fungus. Three distinctive forms predominate: elongated branched septate filaments, large chlamydospores and smaller, elliptical yeast-like single cells. Each of the three forms can be seen both in liquid media and on solid agar surfaces as part of a single colony.
An undesirable characteristic feature of A. pullulans is that it produces a dark pigment which is a melanin-like compound, and appears dark green to black in color.
The pigment contaminates the pullulan due to coprecipitation during recovery of the product. This pigmentation requires that the pullulan be subjected to a multistep decoloration treatment with activated charcoal followed by filtration to remove the pigment before the pullulan can be used. This understandably increases the cost and complexity of manufacture of products from pullulan. See U.S. Pat. Nos. 3,959,009 and 4,004,977.
Another undesirable characteristic of A. pullulans is that as the submerged growth of the organism in a fermentation progresses, the culture viscosity decreases due to a decrease in the average molecular weight of the accumulated extracellular pullulan. (Catley, B. J., 1970, FEBS Letters 10:190-193; Kaplan, D. L., B. J. Wiley, S. Arcidiacono, J. Mayer, and S. Sousa, 1987, Materials Biotechnology Symposium Proceedings, U.S. Army, Natick: 149-173).
U.S. Pat. No. 3,912,591 discloses that the initial culture conditions, e.g., pH, phosphate concentration, carbon source, inoculum size and time of harvest, affect the average molecular size of the polymer and yield of pullulan product. This patent further discloses that increasing the initial pH to 7 or higher results in a decrease in the molecular weight of the pullulan product.